SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.


INTRODUCTION
The global pandemic of COVID-19 has fostered the mutation of SARS-CoV-2 into a number of variants of concern (VOCs). Despite widespread vaccination, the Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs have caused a massive increase in COVID-19 cases and deaths worldwide. Several mutations in the spike proteins of the two VOCs, particularly L452R and P681R in Delta and E484A and Q498R in Omicron variants, have been associated with enhanced viral transmission and high resistance to antibody neutralization (Saito et al., 2022;Sharma et al., 2022). However, the susceptibility of macrophages to these recently emerged VOCs, which are more infectious and could potentially infect cells even more efficiently than the original variant, remains unknown.
SARS-CoV-2 uses the spike protein to bind to ACE2 receptor on the cell surface to undergo cell entry (Hoffmann et al., 2020;Shang et al., 2020). Macrophages have been reported to express ACE2 on the cell surface (Keidar et al., 2005;Song et al., 2020), suggesting that SARS-CoV-2 could infect and replicate in macrophages via an ACE2dependent pathway. However, other studies using single-cell RNA sequencing reported undetectable expression of ACE2 in macrophages (Singh, Bansal & Feschotte, 2020;Liu et al., 2020). Alternatively, infection of macrophages with SARS-CoV-2 could occur by phagocytosis of virions, followed by escape of the virus from the endolysosomal system via an ACE2-independent pathway (Lv et al., 2021). In addition, some studies suggest that alveolar macrophages can support the replication and release of SARS- CoV-2 (Grant et al., 2021;Lv et al., 2021), while others have demonstrated the abortive SARS-CoV-2 infection of macrophages (Niles et al., 2021;Thorne et al., 2021;Zhang et al., 2022). Therefore, it is unknown whether SARS-CoV-2 can replicate in macrophages.
In the lung, SARS-CoV-2 primarily targets and replicates in the alveolar epithelial type II cells lining the alveoli, where macrophages constitute the vast majority of resident immune cells (Lamers & Haagmans, 2022). As innate immune cells, macrophages engulf viral particles and release cytokines to control viral infection. However, whether SARS-CoV-2 could infect macrophages and hijack their cellular machinery as a reservoir to spread its viral progenies to extrapulmonary tissues and organs remains poorly understood. Only a few approaches are available to investigate macrophage susceptibility to SARS-CoV-2 infection, making it difficult to fully understand SARS-CoV-2 pathogenesis.
Derivation of macrophages from hiPSCs provides a new opportunity to study the role of macrophages in SARS-CoV-2 pathogenesis. Unlike human primary macrophages, which are typically obtained in non-uniform batches with limited cell number due to donor variability, iM can be generated in large uniform batches with stable genotype and function (Wilgenburg et al., 2013). Rather than tissue-resident macrophages, iM more closely resemble blood monocyte-derived macrophages (Wilgenburg et al., 2013), which are recruited to the lung during infection. We hypothesized that iM could also become susceptible to infection with two distinct SARS-CoV-2 VOCs: Delta and Omicron, possibly eliciting gene expression of proinflammatory cytokines. Here, we generated and characterized iM to investigate the susceptibility of iM to Delta and Omicron variants as well as their gene expression profiles of proinflammatory cytokines during infection. In contrast to Omicron, Delta variant replicates and causes syncytia formation in iM , suggesting that Delta variant might be able to enter host cells in an ACE2-independent manner and exhibit increased fusogenicity (Saito et al., 2022).

Infection of viruses
iM were infected with SARS-CoV-2 Delta or Omicron variant at a multiplicity of infection (MOI) of 0.1 in RPMI-1640 (Cat# 31800022; Gibco, Waltham, MA, USA) without serum. Mock-infected cells were used as controls. Cells were adsorbed with virus for 2 h, washed with PBS, replenished with RPMI-1640 supplemented with 0.05% TrypLE Select, and incubated at 37 • C/5% CO 2 . Cell supernatants were harvested at 0, 24, 48 and 72 h post-infection (hpi) for virus titration. Cells were subjected to immunofluorescence assay to determine the expression of SARS-CoV-2 N protein.

Immunofluorescence assay of viral proteins
Cells were fixed and permeabilized with 80% acetone for 15 min and blocked with 1% bovine serum albumin (BSA; Cat# A8412; Sigma, St. Louis, MO, USA) in PBST (PBS + 0.1%Tween 20) for at least 30 min. For SARS-CoV-2 detection, cells were stained with primary mouse anti-SARS-CoV-2 N antibodies (1:1000 dilution; in-house) overnight at 4 • C and washed three times with PBS. Cells were then stained with secondary anti-mouse IgG antibodies conjugated with Alexa Fluor 568 (1:1000 dilution; Cat# A11004; Invitrogen, Waltham, MA, USA) and phalloidin conjugated with Alexa Fluor 488 (1:400 dilution; Cat# A12379; Invitrogen, Waltham, MA, USA) in the dark for at least 1 h at room temperature and washed three times with PBS.

Flow cytometry
For surface marker analysis, macrophages were harvested with enzyme-free cell dissociation buffer (Cat# 13151014; Gibco, Waltham, MA, USA). Cells were resuspended in ice-cold FACS buffer (consisting of PBS and 0.5-1% BSA) at a concentration of 1-5 × 10 6 cells/mL. To block non-specific binding of the primary antibodies, Fc receptor blocking antibody (Cat# 422301; BioLegend, San Diego, CA, USA) was added to the cell suspension. Cells were incubated with the conjugated primary antibodies or the isotype-matched control in the dark for at least 30 min at 4 • C. Cells were washed three times with ice-cold FACS buffer before analyzing with a FACSAria Fusion flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo (version 10, Ashland, OR).
The following antibodies (all purchased from Invitrogen, Waltham, MA, USA) were used for staining surface markers

Phagocytosis assay
Macrophage progenitors were cultured for 7 days in macrophage medium in a 96-well plate at a density of 1 × 10 5 cells/well. Mature iM were incubated with or without reconstituted pHrodo Green Zymosan Bioparticles (Cat# P35365; Invitrogen, Waltham, MA, USA) at 37 • C for 2 h. Cells were harvested and analyzed by flow cytometry. Zymosan-free cells were used as negative controls to set a threshold for measuring the percentage of positive cells (Wilgenburg et al., 2013).

Real-time quantitative reverse transcription PCR (RT-qPCR)
For human gene expression, total RNA was extracted from cell lysates using the GeneJET RNA Purification Kit (Cat# K0732; Thermo Fisher, Waltham, MA, USA). RT-qPCR was performed using the iTaq Universal SYBR Green One-Step Kit (Cat# 10032048; Bio-Rad, Hercules, CA, USA) on a CFX Opus 96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Primers for ACE2, CD86, IL-1β, IL-6, IL-8, IL-18, TNF-α, CCL2, IFN-α, and GAPDH are described in Table S1. Cycling conditions were as follows: 1 cycle of 50 • C for 20 min, 1 cycle of 95 • C for 5 min; 40 cycles of 95 • C for 10 s and 58 • C for 30 s. Fluorescence signals were detected at the end of each 58 • C step. The dissociation curve was as follows: 1 cycle of 95 • C for 10 s, 65 • C for 5 s with a temperature increment of 0.5 • C to 95 • C. Ct values and dissociation curves were analyzed using CFX Maestro Software (Bio-Rad; Hercules, CA, USA, version 2.0). Gene expressions were normalized against GAPDH and compared with 293T/17 cells for the ACE2 gene or mock infection for cytokine genes using the 2 − CT method (Schmittgen & Livak, 2008). With the exception of ACE2 and CD86 gene expression, all other samples were measured in triplicate.

Statistical analysis
All statistical tests were performed with GraphPad Prism 9 (San Diego, CA). One-way ANOVA with Tukey's post-hoc tests were used for multiple comparisons between experimental groups. Data are presented as mean ± SD. The P value < 0.05 was considered statistically significant.

Characterization of hiPSC-derived macrophages (iM )
We generated iM using previously published protocols (Figs. 1A-1B) (Wilgenburg et al., 2013;Gutbier et al., 2020). Differentiated iM displayed round, oval, and spindle shapes (Fig. 1B), which are typical of macrophage morphology (Rey-Giraud, Hafner & Ries, 2012). Flow cytometry analysis of cell surface marker expression revealed that iM expressed the monocyte/macrophage lineage markers CD11b, CD14, CD16, CD86 and CD163 ( Fig. 2A). To assess phagocytic activity, iM were incubated with pH-sensitive zymosan particles that fluoresce at an acidic pH typically found in phagosomes. After 2 h of zymosan incubation, the majority (>90%) of iM engulfed the particles (Figs. 2B-2C), confirming that the cells were functional with their phagocytic activity. Taken together, the iM generated in this study exhibit key characteristics of macrophages in terms of their morphology, surface marker expression, and phagocytic activity.

iM are susceptible to SARS-CoV-2 infection
Given that SARS-CoV-2 can infect mouse and human macrophages via ACE2-independent pathways (Lv et al., 2021;Jalloh et al., 2022), we first sought to investigate the susceptibility of iM to SARS-CoV-2 infection. Under bright-field microscopy, we infected iM with SARS-CoV-2 (Delta or Omicron variants) at an MOI of 0.1 and found no apparent signs of CPE at 72 hpi. In contrast, an immunofluorescence experiment revealed the presence of SARS-CoV-2 N protein in the cytoplasm of iM infected with both Delta and Omicron ( Fig. 3A; Fig. S1). SARS-CoV-2 N protein was detectable in 11.5% of Delta-infected iM but only in 3.6% of cells infected with Omicron (Fig. 3B). Of note, we observed clear signs of cell-cell fusion or syncytia formation only in Delta-infected iM ( Fig. 3A; Fig. S1). It is worth noting that, despite the signs of viral entry, RT-qPCR and immunoblot analysis revealed undetectable mRNA and protein expression of ACE2 in iM (Figs. S2A-Figs. S2B; Table S2). Viral replication was further examined by titrating infectious virions in iM supernatants collected up to 72 hpi on A549-ACE2 cells using the TCID 50 assay (Fig.   Thaweerattanasinp et al. (2023) Table S3). Replication of the Delta variant in iM was productive within the first 48 hpi (Fig. 3C). Replication kinetics gradually increased from 8.73 × 10 TCID 50 /mL at 0 hpi to 3.58 ×10 2 and 8.16 ×10 2 TCID 50 /mL at 24 and 48 hpi, respectively (Fig. 3C). Later, however, viral titers dropped to 3.68 × 10 TCID 50 /mL at 72 hpi (Fig. 3C). In contrast, Omicron replication in iM was abortive, as we found no viral titer of the Omicron variant at any time point (Fig. 3C). Taken together, these results suggest that SARS-CoV-2 Delta variant replicates in iM in an ACE2-independent manner and induces syncytia formation.

iM show moderate expression of proinflammatory cytokine genes in response to SARS-CoV-2 infection
We further investigated whether SARS-CoV-2 infection could activate iM to overexpress proinflammatory cytokine genes, leading to overproduction of proinflammatory cytokines or cytokine storms as observed in severe COVID-19 patients (Blanco-Melo et al., 2020;Hadjadj et al., 2020;Grant et al., 2021). To this end, we examined mRNA expression of proinflammatory and antiviral cytokines in iM infected with SARS-CoV-2 (Delta or Omicron) at 24, 48, and 72 hpi as well as in iM polarized to the proinflammatory M1 phenotype by LPS and IFN-γ . After normalization to GAPDH using the 2 − CT method (Schmittgen & Livak, 2008), the results are expressed as a fold change in cytokine gene expression compared to mock infection ( Fig. 4; Table S4). Although the infection rates of SARS-CoV-2 in iM were approximately 11% and 4% for Delta and Omicron,  (Fig. 3B), the infection did activate iM to upregulate mRNA expression of the M1 phenotype marker CD86 ( Fig. S4; Table S5). (LPS) and interferon-gamma(IFN-γ ). Total RNA was extracted from cell lysates at 24 h after LPS/IFN-γ treatment or at 24, 48, and 72 hpi for SARS-CoV-2 infection. Gene expressions were quantified in triplicate by RT-qPCR. Data are expressed as fold change in cytokine gene expression compared with mock infection after normalization to GAPDH using 2 − CT method. Data were shown as means ± SD (n = 3 in each group). One-way ANOVA with Tukey's post hoc test on the log transformation of gene expression fold change.
These results suggest that SARS-CoV-2 infection triggers moderate gene expression of proinflammatory cytokines in iM at the transcriptional level. However, LPS/IFN-γ polarization causes significant transcriptional activation of proinflammatory cytokine genes in iM . Thus, it is unlikely that a direct interaction between SARS-CoV-2 and macrophages is the main cause of increased proinflammatory cytokine production or cytokine storm.

DISCUSSION
In the alveoli, tissue-resident alveolar macrophages serve as sentinels that provide immune surveillance and regulate tissue homeostasis (Kosyreva et al., 2021). Because SARS-CoV-2 can be transmitted via the respiratory tract, tissue-derived and recruited alveolar macrophages are thought to be the first immune cells to encounter the virus in the alveoli. However, isolating human alveolar macrophages for an in vitro study is a difficult task that can only be performed by trained clinicians performing bronchoscopy on sedated humans (Collins et al., 2014). Alternatively, monocyte-derived macrophages from blood samples could be used to investigate the mechanisms of SARS-CoV-2 pathogenesis. However, due to donor heterogeneity, both sources of human primary macrophages provide relatively limited numbers of cells from each donor and inconsistent batches of macrophages. In this study, we successfully generated hiPSC-derived macrophages (iM ) that closely resemble recruited macrophages from blood monocytes (Wilgenburg et al., 2013), to investigate the susceptibility of iM to SARS-CoV-2 Delta and Omicron variants as well as their gene expression profiles of proinflammatory cytokines during infection. Differentiated iM displayed the typical macrophage morphology (Fig. 1) (Rey-Giraud, Hafner & Ries, 2012), expression of macrophage-specific markers ( Fig. 2A) and phagocytic activity (Figs. 2B-2C), confirming the phenotypic and functional macrophage properties of iM .
Although alveolar macrophages are thought to be the first to recognize and interact with SARS-CoV-2 in the alveoli, the role of macrophages in SARS-CoV-2 infection is still largely unknown. Previously, SARS-CoV-2 was shown to infect mouse alveolar macrophages via an ACE2-independent mechanism (Lv et al., 2021). Notably, SARS-CoV-2 was phagocytosed by M1-polarized macrophages, but the virus eventually escaped from the endosome to initiate viral replication in the cytoplasm (Lv et al., 2021). SARS-CoV-2 VOCs, including Delta and Omicron, are associated with increased transmissibility and infectivity (Harvey et al., 2021;Saberiyan et al., 2022). However, it is unclear how the increased characteristics of Delta and Omicron variants alter their interaction with macrophages. In the present study, we found SARS-CoV-2 N protein expression in 11.5% and 3.6% of iM infected with Delta and Omicron variants, respectively (Figs. 3A-3B). However, only Delta infection of iM resulted in the release of infectious virions during the first 48 h (Fig. 3C). This conclusion is inconsistent with previous studies showing that using at least an MOI of 0.1, infection of macrophages with SARS-CoV-2 is abortive (Niles et al., 2021;Jalloh et al., 2022;Zhang et al., 2022). However, since most of these studies used old SARS-CoV-2 isolates for viral infection, the results are likely to be different. Another point worth noting is that iM do not express detectable levels of ACE2 (Fig. S2), making it unlikely that SARS-CoV-2 infection in this cell type is dependent on ACE2 receptor. Surprisingly, Delta infection of iM induced the formation of syncytia in iM ( Fig. 3A; Fig. S1). This observation points to the possibility that syncytia formation may increase cell-to-cell transmission of the SARS-CoV-2 Delta variant in nearby macrophages, allowing the virus to avoid neutralizing antibodies in the extracellular space (e.g., alveolar space) that may prevent cell-free infection (Rajah et al., 2022). Other respiratory viruses, such as measles, influenza, and respiratory syncytial virus, also utilize syncytia, formation for more efficient and rapid viral dissemination (Cifuentes-Muñoz, Dutch & Cattaneo, 2018;Rajah et al., 2022). However, because Omicron variant could infect significantly fewer cells than Delta (Fig.  3B), it is possible that replication and syncytia formation of Omicron in iM would be less likely to be observed. Consequently, macrophages could serve as a viral reservoir for systemic dissemination of the SARS-CoV-2 Delta variant via syncytia formation. However, further research is needed to understand the molecular mechanisms and potential functions of syncytia formation in SARS-CoV-2-infected macrophages.
The immune responses of alveolar macrophages, which are constantly exposed to the outside atmosphere, must be tightly regulated to fight viral infections while minimizing tissue damage and maintaining normal pulmonary function (Divangahi, King & Pernet, 2015). Alveolar macrophages are the major producers of type I IFNs in response to viral infections in the lung (Kumagai et al., 2007;Divangahi, King & Pernet, 2015). However, SARS-CoV-2 infection has been shown to suppress type I IFN antiviral responses (e.g., IFN-α and IFN-β) and elevate the production of inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) in blood and lung samples from COVID-19 patients (Blanco-Melo et al., 2020;Hadjadj et al., 2020;Grant et al., 2021). How SARS-CoV-2 infection affects alveolar macrophage cytokine responses remains largely unclear. In the present study, SARS-CoV-2 infection did not strongly upregulate mRNA expression of proinflammatory and antiviral cytokines in iM (Fig. 4). However, SARS-CoV-2-infected iM increasingly expressed most cytokine mRNAs over time from 24 to 72 hpi during infection (Figs. 4A-4B, 4D, 4F-4G). At 24 hpi, Omicron infection induced significantly higher IL-8 and IFN-α mRNA expression in iM than Delta infection (Figs. 4C, 4G), which could in part contribute to the observed differences in viral replication and syncytia formation between the two variants. However, total proinflammatory cytokine gene expression in response to SARS-CoV-2 infection was at a significantly lower level than LPS/IFN-γ polarization of iM (Figs. 4A-4C, 4E-4F). Moreover, SARS-CoV-2 infection barely triggered antiviral type I IFN-α gene expression in iM (Fig. 4G). These results are consistent with previous studies reporting moderate proinflammatory and antiviral cytokine responses following SARS-CoV-2 infection of primary macrophages (Niles et al., 2021;Thorne et al., 2021;Zhang et al., 2022). Therefore, it is unlikely that direct interaction between SARS-CoV-2 and macrophages is the main reason for the excessive production of proinflammatory cytokines or cytokine storm during early infection. Instead, secreted inflammatory mediators from infected lung epithelial cells may primarily drive macrophages to exacerbate inflammatory responses during the later stages of SARS-CoV-2 infection (Thorne et al., 2021;Zhang et al., 2022). However, without direct measurement of cytokine levels in the cell culture supernatants in this study, we cannot rule out the possibilities of robust proinflammatory cytokine release after SARS-CoV-2 infection of iM and differential cytokine response profiles between Delta and Omicron infection.

CONCLUSIONS
We generated hiPSC-derived macrophages and demonstrated that they are susceptible to productive infection with SARS-CoV-2 Delta variant, probably via an ACE2-independent pathway. The Delta variant also induces syncytia formation in these immune cells, supporting the enhanced fusogenicity of this SARS-CoV-2 variant. However, SARS-CoV-2 infection triggers only moderate gene expression of proinflammatory cytokines in macrophages. The findings suggest that other exogenous stimuli may be the main cause of excessive cytokine production in macrophages during the early phase of SARS-CoV-2 infection. Further in vivo studies are needed to elucidate the molecular mechanisms of the SARS-CoV-2-induced cytokine storm in the lung and systemic inflammation in multiple organs.